This invention relates to a method for treating mammalian blood and plasma with a commercially viable bacterial heparinase preparation to eliminate the interference of normal blood function resulting from the presence of heparin.
Heparin is a sulfated glycosaminoglycan with a backbone comprised of alternating hexuronic, either L-iduronic or D-glucuronic and D-glycocyamine residues joined in alternating 1, 4 linkages. The heterologous nature of heparin is due to the varying degree and location of sulfate substitution on these residues giving rise to at least ten different monosaccharide building blocks within the polymer, as reported by Lindahl, et al., Biosynthesis of heparin TIBS 11 (5): 221-225 (1986). The heterogeneity and high degree of sulfate substitution, greater than 2.6 sulfates per disaccharide unit, gives heparin a high protein binding capacity resulting in the inhibition or activation of several enzyme systems (Sakamoto and Sakamoto, "Heparin and bone metabolism: Effects of heparin on bone collagenase release and activity and an application of heparinsepharose affinity chromatography for in vitro study of bone resorption" in Chemistry and Biology of Heparin (Elsevier/North Holland Press, Amsterdam 1981)). Protein-heparin associations are most frequently due to electrostatic interactions although associations due to tertiary and secondary structure interactions corresponding with specific oligosaccharide sequence regions have also been observed. The most thoroughly studied sequence specific interaction is the stabilization of the antithrombin 3 (AT III)--thrombin complex which results in the inhibition of coagulation, as discussed by Rosenberg and Damus, J. Biol. Chem. 248:6490-6505 (1973).
Heparin is widely used as an anticoagulant in conjunction with invasive surgical procedures and dialysis procedures, to prevent clotting in intravenous lines and in the treatment of thrombolytic disorders. 5-10% of all hospitalized patients have heparin in their blood. Recently, low molecular weight heparins, chemical derivatives of native heparin, have been investigated as potential anticoagulants where the primary mechanism of action is the inhibition of factor Xa, as reported by Choay, et al., Thrombosis Res. 11:240 (1980).
Patients receiving heparin therapy or who have been exposed to heparin through intravenous lines are frequently tested by a variety of means for the assessment of their hematological status or to monitor heparin therapy, itself. For example, activated clotting time assays are performed at 20 minute intervals during extracorporeal procedures to ensure adequate heparinization and the prevention of contact activated clotting. Heparin interferes with several routine hematological analyses. The presence of heparin in the bloodstream will prevent the identification of coagulopathies by standard coagulation assays which rely on clotting as the endpoint. These include activated partial thromboplastin time, prothrombin time, factor compliment assays and activated clotting time. Heparin's electrostatic interactions with essential components of other tests, such as the polylysine substrate in the fibrinolysis assay, causes similar interference problems.
Several approaches have been attempted to circumvent the heparin interference problem. Ion exchange resins have been used to adsorb heparin from the sample prior to testing, as described by Cumming, et al., Thrombosis Res. 41:43-56 (1986). This method is non-specific, removing coagulation factors and other blood proteins in addition to heparin, thereby influencing the test result. The technique is also time consuming and cannot be readily used as a STAT test during surgery where rapid data acquisition is necessary.
Protamine sulfate has been used to neutralize heparin by electrostatic interaction and precipitation, as also reported by Cumming, et al., (1986). The protamine-heparin reaction is stoichiometric and requires an accurate titration to prevent adverse effects resulting from incomplete neutralization or from protamine sulfate's own anticoagulant properties. This method is cumbersome, prone to error and requires large sample volumes for accurate titration measurements. Protamine sulfate is not able to neutralize the anticoagulant effect of low molecular weight heparins.
Another approach is to supplement inhibited thrombin with additional thrombin or a substitute enzyme, such as reptilase, capable of catalyzing a similar reaction, as described by Funk, et al., Brit. J. Haematol. 21:43-52 (1971). This method may be successful for assays intended to monitor events subsequent to fibrin formation but is unsuitable for detecting coagulopathies in the coagulation pathway prior to the thrombin catalyzed reaction. Furthermore, heparin's effects on other components such as platelets, cannot be circumvented by these replacement enzymes.
The most desirable resolution to the heparin interference problem would be a method that could expeditiously and specifically remove heparin from blood samples immediately before the onset of the test. The additive used to accomplish this must function over a broad range of conditions. Heparin should be neutralized, nearly instantaneously, while the reagent, itself, should not impart any effects on blood components over a lengthy exposure period. A reagent demonstrating these characteristics could be used in conjunction with ACT assays which are performed immediately after sample procurement and on samples examined in the hematology laboratory which are liable to sit on the bench for up to one hour before processing. The reagent must function over a broad temperature range, 2.degree. to 37.degree. C., to accommodate samples including: those stored on ice in the hematology laboratory, samples taken from patients undergoing cardiovascular surgery whose blood temperature is maintained at 30.degree. C. and samples from patients undergoing procedures such as dialysis which are carried out at normal body temperatures, 37.degree. C. The heparin neutralizing reagent should also be concentration independent such that a single does of the reagent will effectively neutralize a broad range of heparin concentrations exceeding clinically used quantities (up to 0.3 IU/ml for thrombolytic therapy, up to 1.5 IU/ml for dialysis therapy, and up to 6 IU/ml for cardiovascular surgery). Furthermore, the treated samples containing heparin should give a result identical to untreated samples that have not been exposed to heparin. A candidate reagent would be a degradative glucanase enzyme specific for heparin which had no other effect on the sample as measured by the intended test.
Hutt and Kingdon, J. Lab. Clin. Med. 79:1027 (1972), attempted to use a heparinase from Flavobacterium heparinum to treat plasma samples prior to performing PTT analysis. The authors noted the requirement for purifying the heparinase from crude extracts of Flavobacterium heparinum to reduce an interference effect from the bacterial source. However, their data indicated their inability to completely remove the interfering moiety. A protein preparation containing 0.008 IU heparinase was insufficient in neutralizing 0.1 IU/ml heparin while a preparation containing 0.04 IU heparinase caused an extended PTT time. The success of this preparation relies on an accurate titration of the enzyme and would be limited in the amount of heparin that could be neutralized.
U.S. Pat. No. 4,795,703 to Folkman, et al, describes using heparinase from Flavobacterium heparinum to develop a method for making quantitative heparin determinations in whole human blood samples using an activated clotting time assay. They were similarly unable to completely remove an interfering moiety from their enzyme preparations. Heparinase treated samples originally containing heparin demonstrated ACT times 16% longer than untreated samples that had not been exposed to heparin. This effect was not detrimental to their process as quantitative heparin determinations relied on comparing test results to a standard curve which presumably took into account the influence of the enzyme preparation on the test result.
Neither group identified the Flavobacterium heparinum moiety responsible for influencing the test results nor demonstrated its resolution from the active Flavobacterium heparinum component responsible for heparin neutralization. The authors of these papers could not differentiate whether their preparations contained an additional molecule from Flavobacterium heparinum that acted as an anticoagulant or whether heparinase, itself, has anticoagulant properties when used in excess or in combination with heparin. The recent characterization of myxalin, a glycoprotein from a Gram negative bacterial source, demonstrates that it possesses anticoagulant properties associated with its carbohydrate moiety, as reported by Akoum, et al. Thrombosis Res. 60:9-18 (1990). Evidence strongly indicates that heparinase is also a glycoprotein, thereby substantiating its potential to have similar characteristics.
Bohmer, et al., Thrombosis Res. 60: 331-335 (1990) have used heparinase III from an undetermined, unavailable bacterial source to neutralize heparin prior to aPTT and PT assays and examined its potential to neutralize heparin's inhibition of thrombin mediated fibrinogen degradation. Incubations of the enzyme with citrated plasma at 37.degree. C. for five minutes and with whole blood at 37.degree. C. for 15 minutes were used to achieve the desired heparin neutralization. The optimal activity profile of heparinase III, pH 7.6, [NaCl]=0.03 and T=45.degree. C. (16), may have necessitated the inclusion of an incubation period in the reported protocol.
Neither of these three groups presented evidence for the stability of their heparinase formulation, an essential component of a clinical product.
One of heparin's primary therapeutic uses is to prevent coagulation during cardiovascular surgery while the patient's blood is circulating through an extracorporeal circuit. Approximately ten times the normal thrombolytic dose of heparin is used in this application, requiring post-operative neutralization by the administration of protamine. Since protamine reversal is associated with several complications and requires titration, an alternative approach to the heparin/protamine control of hemostasis is desirable. One method may be to replace protamine reversal with heparinase, directly injected into the patient after bypass.
Langer and co-workers investigated the in vivo effects of heparinase in animal models, as reported by Klein, et al., J. Lab. Clin. Med. 102:828-837 (1982) and Langer, et al., Trans. Am. Soc. Artific. Intern. Organs 28:387-390 (1982). Heparinase with a specific activity of 0.58 IU/mg was injected into rabbits that had received heparin and aPTT assays were used to measure coagulation over time. An accelerated heparin clearance was noted as compared to rabbits which did not receive heparinase. However, coagulation times were still three times baseline, 15 minutes after the heparinase injection and returned to baseline only 1 h later, the same period of time for the control animal which did not receive a heparinase injection. Langer was able to achieve better reversal by pumping blood through an immobilized heparinase reactor. Following this result, the Langer group abandoned the idea of direct heparinase injection and focused on developing the extracorporeal use of heparinase to replace protamine.
It is therefore an object of the present invention to provide a heparinase preparation which can be used to quickly and completely neutralize heparin in a wide range of concentrations, both in vitro and in vivo.
It is a further object of the present invention to provide a heparinase preparation which is free of any contaminants altering coagulation times.
It is a still further object of the present invention to provide a heparinase preparation which is stable for an extended period of time at room temperature.